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Expression in Escherichia Coli, Refolding, and Purification of Human Procathepsin K, an Osteoclast-Specific Protease

Expression in Escherichia Coli, Refolding, and Purification of Human Procathepsin K, an Osteoclast-Specific Protease

K J D'alessio, M S McQueney, K A Brun, M J Orsini, C M Debouck

Protein Expr Purif. 1999 Mar;15(2):213-20.

PMID: 10049678

Abstract:

We have constructed and optimized a high yielding Escherichia coli expression system to produce glycosylation-free human procathepsin K and have developed conditions for refolding this enzyme. Recombinant human procathepsin K (EC 3.4.22.38) was expressed in E. coli, refolded from inclusion bodies, and further purified by Superdex 75 size-exclusion chromatography. Purified procathepsin K had a [MH]+ of 35,063 Da which is in agreement with the predicted mass of the construct. Amino-terminal sequence analysis matched the predicted sequence with no secondary sequence detected. Purified procathepsin K activated under autocatalytic conditions to a final specific activity of 23 micromol 7-amido-4-methylcoumarin liberated/min/mg of enzyme using the fluorescent peptide substrate benzyloxycarbonyl-phenylalanine-arginine-7-amido-4-methylcoumarin. This expression and refolding procedure yielded 50 mg of purified, glycosylation-free human procathepsin K from 1 liter of E. coli cell culture and enabled the determination of the structure of human procathepsin K at 2.6 A resolution.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42412613	Procathepsin K human			Price

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